Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy

• DOI: 10.1073/pnas.1213676110
• Publication Date: 2013-02-20T06:56:59Z
• Authors: Graeme A. King; Peter Gross; Ulrich Bockelmann; Mauro Modesti; Gijs J. L. Wuite; Erwin J. G. Peterman
• Keywords: DNA origami

Mechanical stress plays a key role in many genomic processes, such as DNA replication and transcription. The ability to predict the response of double-stranded (ds) tension is cornerstone understanding mechanics. It widely appreciated that torsionally relaxed dsDNA exhibits structural transition at forces ∼65 pN, known overstretching, whereby contour length molecule increases by ∼70%. Despite extensive investigation, changes occurring during overstretching are still generating considerable debate. Three mechanisms have been proposed account for increase overstretching: strand unpeeling, localized base-pair breaking (yielding melting bubbles), formation S-DNA (strand unwinding, while base pairing maintained). Here we show, using combination fluorescence microscopy optical tweezers, all three structures can exist, uniting often contradictory dogmas overstretching. We visualize distinguish unpeeling melting-bubble an appropriate fluorescently labeled proteins, whereas remaining B-form accounted specific fluorescent molecular markers. Regions associated with domains where probes do not bind. demonstrate balance between overstretched governed both topology local stability. These findings enhance our knowledge mechanics stability, which fundamental importance how proteins modify physical state DNA.